The present invention generally relates to methods for recovering polyhydroxyalkanoates (xe2x80x9cPHAsxe2x80x9d) from microbial or plant biomass. An improved understanding of the PHA biosynthetic pathways has allowed for the use of microbial organisms, both natural and recombinant, as well as plant cells, to produce significant quantities of PHA. However, difficulties remain in developing efficient and cost-effective recovery of the PHA at a useful levels of quality and purity from these biological source materials. Previous methods for isolating and purifying PHAs from biomass have included, for example, aqueous routes as well as organic solvent routes.
For example, U.S. Pat. No. 5,364,778 to Byrom discloses an aqueous method wherein biomass comprising PHA is maintained as an aqueous slurry in which the PHA is generally insoluble. The slurry is subjected to various treatments designed to digest, degrade, or otherwise make water-soluble the non-PHA biomass. This solubilized biomass then is removed from the slurry by centrifugation, filtration, or other means. Aqueous-based routes, however, generally have certain disadvantages, particularly which applied to large scale processing. Examples of these disadvantages include (a) effective purification is made more difficult because many impurities, including some surfactants useful for the solubilizing treatments, may be tightly adsorbed to the surface of the PHA particles; (b) many volumes (i.e. large quantities) of wash water may be required by the process, creating used wash water and its attendant disposal difficulties; (c) multiple solubilizing treatments may be required to obtain high purity PHA; (d) drying of the water-based PHA slurry may be time-consuming and costly; (e) PHA particles may cause extensive fouling of filtration membranes, centrifuges, and other process equipment; and (f) solubilizing treatments may require expensive reagents and lengthy process times and/or high temperatures to be effective.
Examples of organic solvent-based methods processes are disclosed in U.S. Pat. No. 4,101,533 to Lafferty et al. and U.S. Pat. No. 5,422,257 to Ohleyer. In these methods, an organic solvent for the PHA contained in a biomass is mixed with the biomass, resulting in the dissolution of the PHA. The organic solution comprising the PHA then is separated from the remaining insoluble biomass by filtration, centrifugation, or other means. The organic solution then is desolventized to recover the PHA. These organic solvent routes suffer disadvantages similar to the disadvantages associated with aqueous routes, including (a) a relatively large volume of solvent is required to completely extract the PHA from biomass; (b) biomass may need to dried prior to solvent extraction, which may be costly and time-consuming; and (c) solvents may co-extract impurities along with the PHA, such as lipids or other hydrophobic biological materials, necessitating further processing of the extract to obtain PHA of satisfactory purity. It would be advantageous to develop improved, more cost-effective processes for recovering PHA from PHA-containing biomass.
It is therefore an object of this invention to provide a method of recovering PHA from PHA-containing biomass using a process that is more simple, relatively faster, uses aqueous and/or organic solvents more efficiently, and possibly yields a more pure PHA product than conventional processes.
It is another object of the present invention to provide a method of recovering PHA from PHA-containing biomass using a process that can be employed economically in a commercial-scale production process.
A method is provided for isolating and purifying PHA from biomass which comprises PHA. The method includes the step of extracting PHA from the biomass using at least one solvent while simultaneously subjecting the biomass to a filtration process to remove cells. In a preferred embodiment of the method, biomass comprising PHA (for example an aqueous slurry of microbial cells obtained from a fermentation process) is directly extracted by diafiltration sing an organic solvent, to obtain PHA.
In a preferred diafiltration process, an aqueous slurry of microbial cells comprising PHA is recirculated through a filtration membrane, wherein the membrane has a pore size sufficiently small to reject individual cells or such aggregates of cells as may exist in the slurry. An outflow of liquid from the filtration membrane occurs under conditions where a pressure drop exists across the filtration membrane. As the liquid is progressively removed from the biomass slurry, an organic solvent, preferably a water-miscible solvent that also is a solvent for the PHA, is added to the biomass slurry. The solvent addition should be made at a rate which approximates the rate of liquid permeation through the filter in order to maintain the volume of the biomass slurry. As the concentration of organic solvent in the slurry increases, various impurities which are insoluble in water become dissolved in the solvent-water mixture and pass through the filter membrane. When the organic solvent concentration reaches a certain level, the PHA becomes soluble and flows through the filtration membrane. The filtrate comprising PHA then is desolventized to recover the polymer.
The method has the advantages that (a) it is not generally necessary to dry the biomass prior to solvent extraction; (b) it is readily possible to fractionate the PHA from other impurities to obtain relatively pure PHA in a single process, because the biomass is subjected to a gradient in solvent concentration; (c) the entire process of extracting and purifying PHA from biomass can be accomplished using a minimum of process stages and equipment; and (d) the method efficiently uses solvents, especially when the biomass slurry is relatively concentrated and when the diafiltration is conducted at a constant volume diafiltration. Furthermore, by using volatile organic solvents, it is relatively easy to desolventize the PHA solutions and to recover and reuse the solvent from the filtrates generated in the diafiltration process.
A method has been developed for isolating and purifying polyhydroxyalkanoates (xe2x80x9cPHAsxe2x80x9d)from biomass comprising PHAs. The method includes the step of extracting PHA from the biomass using at least one solvent while simultaneously subjecting the biomass to a filtration process to remove cells.
1. The PHA-Containing Biomass
The biomass materials are derived from PHA-producing plants or PHA producing microorganisms.
Polymer Compositions
As used herein, xe2x80x9cpolyhydroxyalkanoatexe2x80x9d and xe2x80x9cPHAxe2x80x9d refer to polymers that contain one or more units, for example between 10 and 100,000, and preferably between 100 and 30,000 units of the following formula I:
xe2x80x94OCR1R2(CR3R4)nCOxe2x80x94;
wherein n is an integer, for example between 1 and 15, and in a preferred embodiment, between 1 and 4; and
wherein R1, 2, R3, and R4 independently can be hydrocarbon radicals including long chain hydrocarbon radicals; halo- and hydroxy-substituted radicals; hydroxy radicals; halogen radicals; nitrogen-substituted radicals; oxygen-substituted radicals; and/or hydrogen atoms.
As used herein, the formula xe2x80x94(CR3R4)nxe2x80x94 is defined as including the following formulas:
xe2x80x94CR3R4xe2x80x94(where n=1);
xe2x80x94CR3R4CR3xe2x80x2R4xe2x80x2xe2x80x94(where n=2); and
xe2x80x94CR3R4CR3xe2x80x2R4xe2x80x2CR3xe2x80x3R4xe2x80x3xe2x80x94(where n=3);
wherein R3, R4, R3xe2x80x3, R4xe2x80x3, R3xe2x80x3, and R4xe2x80x3, can be independently hydrocarbon radicals including long chain hydrocarbon radicals; halo- and hydroxy-substituted radicals; hydroxy radicals; halogen radicals; nitrogen-substituted radicals; oxygen-substituted radicals; and/or hydrogen atoms. Thus, formula I includes units derived from 2-hydroxyacids (n=0), 3-hydroxyacids (n=1),4-hydroxyacids (n=2), and 5-hydroxyacids (n=3), and 6-hydroxyacids (n=4).
These units may be the same in a homopolymer, or be more different units, as for example in a copolymer or terpolymer. The polymers typically have a molecular weight over 300, for example between 300 and 107, and in a preferred embodiment 10,000 to 10,000,000 Daltons.
Preferred PHAs include poly-3-hydroxyoctanoate (PHO) or other microbial polyesters comprising hydroxyacids from C6 to C14 hydroxyacids. Other preferred polymers include poly-3-hydroxybutyrate-co-3-hydroxyvalerate, poly-3-hydroxybutyrate-co-3-hydroxypropionate, poly-3-hydroxybutyrate-co-4-hydroxybutyrate, poly-3-hydroxybutyrate-co-4-hydroxyvalerate, poly-3-hydroxybutyrate-co-3-hydroxyhexanoate, poly-3-hydroxybutyrate-co-3-hydroxyoctanoate, poly-4-hydroxybutyrate, poly-3-hydroxypropionate, poly-4-hydroxyvalerate.
Sources of PHA-Containing Biomass
The PHA biomass is typically generated from a fermentation process (wherein the biological source is a microorganism which naturally produces the PHAs or which can be induced to produce the PHAs by manipulation of culture conditions and feedstocks, or microorganisms) or produced in a plant, or plant part, which has been genetically engineered so that it produces PHAs.
(i) Microbial Sources
Methods which can be used for producing PHA polymers from microorganisms which naturally produce polyhydroxyalkanoates are described in U.S. Pat. No. 4,910,145 to Holmes, et al.; Braunegg et. al., J. Biotechnology 65:127-161 (1998).
Methods for producing PHAs in natural or genetically engineered organisms are described in Madison and Huisman, Microbiol. Mol. Biol. Rev. 63:1-53 (1999); Choi and Lee, Appl. Microbiol. Biotechnol. 51:13-21 (1999); Witholt and Kessler, Current Opinion in Biotechnology 10:279-285 (1999); Williams and Peoples, CHEMTECH, 26:38-44 (1996); U.S. Pat. Nos. 5,245,023; 5,250,430; 5,480,794; 5,512,669; 5,534,432 to Peoples and Sinskey; and U.S. Pat. No. 5,563,239 to Hubbs et al. U.S. Pat. No. 5,292,860 to Shiotani et al. describes the manufacture of the PHA copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate. U.S. Pat. No. 5,871,890 to Naylor describes the manufacture of PHAs by fermenting Alcaligenes eutrophus on vegetable oil feedstocks.
(ii) Plant Sources
PHA can be recovered from essentially any plant type, including transgenic plants which offers many advantages for the production of PHAs. Transgenic crop plants for production of PHAs can be obtained using methods available in the art. (U.S. Pat. Nos. 5,245,023 and 5,250,430; 5,502,273; 5,534,432; 5,602,321; 5,610,041; PCT WO, 9100917, WO 9219747, WO 9302187, WO 9302194 and WO 9412014; Poirier et al., 1992 Science 256:520-23, van der Leij and Witholt, 1995, Can. J. Microbiol. 41 (supp):222-38; Nawrath and Poirier, 1996, presented at The International; Symposium on Bacterial Polyhydroxyalkanoates, Eggink et al., eds. Davos Switzerland, August 18-23; Williams and Peoples, 1996, CHEMTECH 26:38-44). Transgenic plant crop production can produce PHA polymers at both a price and a scale that is competitive with petrochemical derived plastics. Transgenic plant derived PHA polymers or their derivatives can be processed and separated from plant biomass in commercially useful forms. The location of the PHA in the plant crop can be varied to maximize the yield of PHA from the plant. For example, the plants can be monocots or dicots and suitable plant source materials can be derived from roots, stems, leaves, flowers, fruits, and seeds.
PHAs can be isolated from plant biomass derived from plants such as soybean, cotton, coconuts, groundnuts, rapeseed, sunflower seed, olive, palm, sesame seed, linseed, castor, safflower seed, tobacco, sugarcane, swithchgrass, and potato. In addition to the PHA polymers, the plant oil in seed crop plants can be isolated and recovered during the processing, as described in PCT WO 97/15681 to Metabolix, Inc. and U.S. Ser. No. 08/548,840, which is incorporated by reference herein. The methods for processing the plant biomass can be tailored based on the properties of the particular PHA polymer or derivative being isolated, and based on the type of plant crop and the plant components being extracted.
III. Process for PHA Recovery from Biomass
The method includes the step of extracting PHA from the biomass using at least one solvent while simultaneously subjecting the biomass to a filtration process to remove cells.
Diafiltration
In a preferred embodiment of the method, biomass comprising PHA (for example an aqueous slurry of microbial cells obtained from a fermentation process) is directly extracted by modification of a typical diafiltration process in which an organic solvent is used instead of an aqueous diluent. Standard diafiltration processes are well known in the art and are described for example by Zeman and Zydney, Microfiltration and Ultrafiltration Principles and Applications, Marcel Dekker, Inc. New York, N.Y. pp. 391-96 (1996). During this modified process, as the concentration of organic solvent increases, the PHA is solubilized and appears in the eluant which is collected. The PHA is then recovered from the eluant by standard procedures including precipitation in a non-solvent, solvent evaporation or stripping to recover the PHA. The solvent containing eluant is retained and the solvent recovered by distillation or other techniques well known in the art.
In a preferred embodiment of the method, biomass comprising PHA (for example an aqueous slurry of microbial cells obtained from a fermentation process) is directly extracted by diafiltration using an organic solvent, to obtain PHA.
The method has the advantages that (a) it is not generally necessary to dry the biomass prior to solvent extraction; (b) it is readily possible to fractionate the PHA from other impurities to obtain relatively pure PHA in a single process, because the biomass is subjected to a gradient in solvent concentration; (c) the entire process of extracting and purifying PHA from biomass can be accomplished using a minimum of process stages and equipment; and (d) the method efficiently uses solvents, especially when the biomass slurry is relatively concentrated and when the diafiltration is conducted at a constant slurry volume (xe2x80x9cconstant volume diafiltrationxe2x80x9d). Furthermore, by using volatile organic solvents, it is relatively easy to desolventize the PHA solutions and to recover and reuse the solvent from the filtrates generated in the diafiltration process.
In a preferred diafiltration process, an aqueous slurry of microbial cells comprising PHA is recirculated through a filtration membrane, wherein the membrane has a pore size sufficiently small to reject individual cells or such aggregates of cells as may exist in the slurry. An outflow of liquid, the eluant which can be an aqueous solution, an aqueous solution/miscible solvent mixture, or solvent, from the filtration membrane occurs under conditions where a pressure drop exists across the filtration membrane. As the liquid is progressively removed from the biomass slurry, an organic solvent, preferably a water-miscible solvent that also is a solvent for the PHA, is added to the biomass slurry. The solvent addition should be made at a rate which approximates the rate of liquid permeation through the filter in order to maintain the volume of the biomass slurry. As the concentration of organic solvent in the slurry increases, various impurities which are insoluble in water become dissolved in the solvent-water mixture and pass through the filter membrane. When the organic solvent concentration reaches a certain level, the PHA becomes soluble and flows through the filtration membrane. The filtrate comprising PHA then is desolventized to recover the polymer.
Organic Solvents and Solvent Recovery
Solvents suitable for extracting the PHA from the biomass are any water miscible solvent capable of extracting the PHA. It is well known in the art which solvents are suitable for extracting the different PHA polymer compositions as described for example in U.S. Pat. Nos. 5,821,299 and 5,942,597 to Noda; U.S. Pat. No. 6,043,063 to Kurdikar; and PCT WO 97/15681 to Metabolix, Inc., all of which are incorporated herein by reference.
A preferred organic solvent for PHAs such as poly-3-hydroxyoctanoate (PHO) or other microbial polyesters comprising hydroxyacids from C6 to C14 in length is acetone. Acetone is also suitable for extracting poly-3-hydroxybutyrate-co-4-hydroxybutyrate. Other ketones and alcohols, especially alcohols above C2, can be used as described above. For PHO, solubilization of the polyester typically occurs at an acetone concentration from 85-48% in water (volume basis).
Organic solvents useful in the methods described herein include both halogentated and nonhalogentated solvents. Representative examples include solvents selected from cyclic and acyclic (linear and branched) Rxe2x80x2xe2x80x94OH alcohols where Rxe2x80x2xe2x95x90C4-Cl10, cyclic and acyclic Rxe2x80x3xe2x80x94COORxe2x80x2xe2x80x3 esters where Rxe2x80x3xe2x95x90H or C1-C6 and Rxe2x80x2xe2x95x90C4-C10, cyclic and acyclic Rxe2x80x3xe2x80x94COORxe2x80x2xe2x80x3 esters where Rxe2x80x2xe2x95x90H or C1-C6 and Rxe2x80x2xe2x80x3xe2x95x90C1-C7, and wherein at least one oxygen is substituted for at least one carbon in Rxe2x80x3 or Rxe2x80x2xe2x80x3, cyclic and acyclic R1xe2x80x94CONxe2x80x94(R2)2 amides where R1=H or C1-C6 and R2=C1-C6, and cyclic and acyclic R3xe2x80x94COxe2x80x94R4 ketones where R3=C1-C6 and R4=C1-C6.
Specific examples include acetone, butyl acetate, isobutyl acetate, ethyl lactate, isoamyl acetate, benzyl acetate, 2-methoxy ethyl acetate, tetrahydrofurfuryl acetate, propyl propionate, butyl propionate, pentyl propionate, butyl butyrate, isobutyl isobutyrate, ethyl butyrate, ethyl valerate, methyl valerate, benzyl benzoate, methyl benzoate, dimethyl succinate, dimethyl glutarate, dimethyl adipate, isobutyl alcohol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1 butanol, 1-pentanol, 3-pentanol, amyl alcohol, allyl alcohol, hexanol, heptanol, octanol, cyclohexanol, 2-ethylhexanol, tetrahydrofurfuryl alcohol, furfuryl alcohol, benzyl alcohol, 2-furaldehyde, methyl isobutyl ketone, methyl ethyl ketone, g-butyrolactone, methyl n-amyl ketone, 5-methyl-2-hexanone, ethyl benzene, 1,3-dimethoxybenzene, cumene, benzaldehyde, 1,2-propanediol, 1,2-diaminopropane, ethylene glycol diethyl ether, 1,2,3-trimethylbenzene, 1,2,4-trimethylbenzene, 1,3-dioxane, 1,4-dioxane, 1-nitropropane, toluene-2,4-diisocyanate, acetic acid, acrylic acid, acetic anhydride, alpha-methylstyrene, acetophenone, toluene, ethylene glycol diacetate, dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide and propylene carbonate.
Solvents which can be used include solvents or solvent mixtures including chloronated organic solvents such as chloroform, methylene chloride, dichloroethane, trichloroethane, tetrachloroethane and dichloroacetate. For example, hydrocarbon stabilized chloroform can be used. Other solvents which have been used to extract PHAs from microbial sources which may be used include alkyl carbonates, such as propylene carbonate and ethylene carbonate, trifluoroethanol, acetic anhydride, dimethylformamide, ethylacetoacetate, triolein, toluene, dioxane, tetrahydrofuran, diethylether, pyridine, hydroxyacids and alcohols having more than 3 carbon atoms, as well as mixtures thereof
Solvent recovery can be carried out by processes well known to those skilled in the art and includes distillation or extraction into a second solvent or solvent mixture which is not miscible with water and subsequent separation by distillation
Recovery of the PHA From the Eluant or Filtrate
Once the polymer appears in the filtrate or eluant, it is necessary to recover the polymer from the solvent and also to recover the solvent.
Techniques for doing this are also well known in the art and include solvent stripping or evaporation, steam stripping or solvent precipitation with a non-solvent.